ES2587630T3 - Expression vector system comprising two selection markers - Google Patents

Abstract

An expression vector or a combination of at least two expression vectors, comprising at least: (a) a polynucleotide encoding a product of interest or an insertion site to incorporate a polynucleotide encoding a product of interest; (b) a polynucleotide encoding a first selection marker (sm I); (c) a polynucleotide encoding a second selection marker (sm II), which differs from the first selection marker (sm I), wherein the activity of the selection marker (sm I) or (sm II) is at least partially influenced by the activity of the other selection marker and where the selection markers (sm I) and (sm II) are involved in folate metabolism and where the first selection marker (sm I) is a folate transporter and the second selection marker (sm II) is DHFR or a functional variant thereof.

Description

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Host cells survive / proliferate under selective culture conditions that, despite selective culture conditions, can maintain the metabolic process sufficiently active to allow cell growth and survival. Survival / growth is promoted if the host cells express both selection markers (sm I) and (sm II) and, consequently, the expression vectors introduced with high yield. In this way, host cells are selected that also express the product of interest, with high yield.

A "metabolic process" describes in particular a process in the host cell, which is essential for cell viability and / or cell proliferation. Examples of metabolic processes are nucleic acid synthesis or polypeptide synthesis. A "metabolic pathway" refers in particular to a subgroup of a metabolic process and describes a defined series of chemical reactions that occur within a cell. In each route, a main chemical is modified by chemical reactions. A classic example of a metabolic pathway is the synthesis of nucleotides (belonging to the metabolic process of nucleic acid synthesis), in particular the biosynthesis of purine or pyrimidine or the synthesis of amino acids (belonging to the metabolic process of polypeptide synthesis). There are several levels of metabolic pathways, which are often also interdependent and therefore connected. Therefore, these terms should be understood more functionally as individual metabolic pathways and also metabolic processes often overlap.

The first selection marker (sm I) and the second selection marker (sm II) are involved in folate metabolism.

Folate metabolism is important to maintain the cell viability of the host cell and / or for the proliferation of host cells. Therefore, folate metabolism is a suitable functional point for the selection system according to the present invention. Therefore, this metabolic pathway is very suitable for selecting the appropriate selection markers, (sm I) and (sm II) as described herein and suitable selection conditions that allow the selection of host cells expressing said markers. Suitable selection markers involved in the respective metabolic pathways, as well as suitable host cells and suitable selection conditions are known in the prior art and are described below and, therefore, can be used in conjunction with the present invention.

According to one embodiment, the first selection marker (sm I) and / or the second selection marker (sm II) is a eukaryotic selection marker. A "eukaryotic selection marker" allows selection of eukaryotic host cells, comprising, respectively, expressing that selection marker. The eukaryotic selection marker can be a metabolic selection marker and, consequently, a marker that is involved in a process or metabolic pathway of the cell, for example, synthesis of nucleic acids or polypeptides.

The second selection marker (sm II) which is DHFR or a functional variant thereof, is an amplifiable selection marker. An amplifiable selection marker allows the selection of host cells containing the vector and promotes gene amplification.

The first selection marker (sm I) and / or the second selection marker (sm II) can be a catalytic polypeptide or a transporter polypeptide. There are many respective suitable selection markers that can be used in conjunction with the present invention and will be explained in detail below.

The second selection marker (sm II), which is DHFR or a functional variant thereof, is a catalytic polypeptide that processes a substrate that is a compound that is incorporated by the first selection marker (sm I), which is a folate transporter, into the host cell or a subsequent product obtained from said incorporated compound.

Accordingly, the selection marker (sm II) processes a substrate that is imported into the host cell by the activity of the first selection marker (sm I). Said compound that is incorporated into the host cell by the first selection marker (sm I) can also be a precursor to the actual substrate that is processed by the second selection marker (sm II). Therefore, the catalytic polypeptide (sm II) can also process that it is a subsequent product obtained from the compound that is incorporated into the host cell by the activity of the first selection marker (sm I).

Without being bound by theory, it is assumed that the activity of the second selection marker (sm II) depends strongly on the activity of the first selection marker (sm I) under selective culture conditions for both markers. The presence and / or amount of substrate for the second selection marker (sm II) depends at least to some extent on the appropriate expression / activity of the selection marker (sm I). The strong overexpression of the first selection marker (sm I) leads to a higher substrate availability for the second selection marker (sm II). The greater bioavailability of the substrate increases the activity of the second selection marker (for example, the renewal index). However, if the selection marker (sm I) is not

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for the regulated biosynthesis of AMP and GMP. Additionally, 5,10-methylene-THF (5,10-CH2-THF) is another important THF coenzyme that works as a crucial co-factor for the thymidylate synthase (TS) enzyme. Thymidylate synthase (TS) catalyzes the formation of thymidine monophosphate (dTMP) from dUMP using 5,10-methylene-THF (5,10-CH2-THF), thereby providing dihydrofolic acid. Dihydrofolate reductase (DHFR) catalyzes the NADP-dependent reduction of dihydrofolic acid (DHF) to tetrahydrofolic acid (THF). THF is then interconverted to 10-formyl-THF and 5,10-methylene-THF, which are used in de novo biosynthesis of purines and thymidylate, respectively. This reaction is catalyzed by serine hydroxy methyl transferase (SHMT). Dihydrofolic acid (DHF) is, therefore, the byproduct of the catalytic activity of thymidylate synthase (TS) that catalyzes the conversion of dUMP to dTMP in a 5,10-methylene-THF-dependent reaction. Therefore, dihydrofolate reductase (DHFR) is crucial for the recycling of THF co-factors, which are essential for the biosynthesis of the purine and pyrimidine nucleotides that are necessary for DNA replication.

The enzymes described, which are to some extent dependent on folate, are the key mediators of de novo biosynthesis of purine and thymine nucleotides essential for DNA replication.

The second selection marker (sm II) processes a substrate that is a folate, a folate derivative and / or a product that can be obtained by folate processing, such as DHF or THF or a functional variant or a derivative of the previous. The respective substrates are important for the production of nucleic acids. Said second selection marker (sm II) is or comprises dihydrofolate reductase (DHFR). This is particularly suitable since the selection marker (sm I) is a folate transporter.

Several suitable DHFR enzymes and, in accordance with the same, genes, which can be used in conjunction with the present invention, are known in the prior art. The DHFR may be a wild-type DHFR or a functional variant or derivative thereof. The term a "variant" or "derivative" includes DHFR enzymes that have one or more exchanges of the amino acid sequences (eg, deletions, substitutions or additions) with respect to the amino acid sequence of the respective DHFR enzyme, fusion proteins , which comprise a DHFR enzyme or a functional fragment thereof and DHFR enzymes that have been modified to provide additional structure and / or function, as well as functional fragments thereof, which still have at least one function of a DHFR enzyme . For example, a DHFR enzyme can be used as a selection marker (sm II) that is, for example, more or less sensitive for anti-folates, such as MTX, than the wild-type DHFR enzyme and / or the enzyme DHFR endogenously expressed by the host cell if expressed. The DHFR enzyme can be derived from any species, provided it is functional within the present invention, that is, compatible with the host cell used. For example, a mutant mouse dihydrofolate reductase (DHFR) with increased MTX resistance has been widely used as a dominant selection marker that greatly improves the acquisition of high level MTX resistance in transfectant cells. Preferably, a DHFR enzyme is used as a selection marker, which is less susceptible to a DHFR inhibitor, such as MTX, than the endogenously expressed DHFR enzyme in a DHFR + (positive) host cell.

According to one embodiment, an intron or fragment thereof is placed at the 3 'end of the open reading frame of the DHFR gene. This has convenient effects on the expression / amplification rate of the construction. The intron used in the DHFR expression cassette leads to a smaller non-functional variant of the DHFR gene (Grillari et al., 2001, J. Biotechnol. 87, 59-65). In this way, the level of expression of the DHFR gene decreases, and, consequently, the rigor of the selection system is further increased. Accordingly, the host cell may comprise an introduced polynucleotide encoding a DHFR enzyme, this polynucleotide comprising an intron that is located in 3 ′ of the sequence encoding DHFR. Alternative methods that make use of an intron to reduce the level of expression of the DHFR gene are described in EP0 724 639 and could also be used.

According to a preferred embodiment, the first selection marker (sm I) is a folic acid receptor and the second selection marker is a variant of the DHFR that is less sensitive to MTX than the wild-type DHFR enzyme and / or DHFR enzyme endogenously expressed by the host cell. This variant of the DHFR preferably also comprises an intron as described above. Particularly preferred is a combination of the respective label in combination with DHFR + (positive) cells.

The teaching of the present invention can be carried out using different embodiments of expression vectors and / or combinations of at least two expression vectors as described herein. The polynucleotide encoding a product of interest or the insertion site to incorporate a respective polynucleotide, the polynucleotide encoding a first selection marker (sm I) and / or the polynucleotide encoding a second selection marker (sm II) and optionally Additional vector elements, such as additional selection markers, can be located on the same expression vectors or on separate expression vectors.

For example, the use of an expression vector, which comprises at least all the decisive elements described above, that is, the polynucleotide encoding the product of interest (or an insertion site for

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cell surface and cells exhibiting high levels of membrane-anchored fusion polypeptide can be selected by flow cytometry, preferably by FACS. In this way, host cells that have a high expression rate are selected. Details and preferred embodiments of this stop codon-based technology are described in WO2005 / 073375 and PCT / EP2009 / 006246. Reference is made to this disclosure.

According to an alternative embodiment, the expression cassette comprises at least:

(to)

the polynucleotide that encodes the product of interest,

(b)

an intron, which comprises a 5 'splice donor site and a 3' splice acceptor site and comprising a translation stop codon within the frame and a polyadenylation signal, and

(C)

a polynucleotide downstream of this intron encoding a membrane anchor and / or a signal for a membrane anchor.

This expression cassette design has the effect that, through transcription and transcription processing, at least two different mature mRNAs (mRNA-POI) and (mRNA-POI-ANCHOR) are obtained from the cassette expression. The translation of the mRNA-POI results in the product of interest. Translation of the mRNA-POI-ANCHOR results in a fusion polypeptide, which comprises the product of interest and a membrane anchor. As a result, this fusion polypeptide is again displayed on the cell surface and cells that exhibit high levels of membrane-anchored fusion polypeptide can be selected by flow cytometry, preferably FACS. In this way, host cells that have a high expression rate are selected. Details and preferred embodiments of this intron-based technology are described in WO2007 / 131774. Reference is made to this disclosure.

According to a preferred embodiment, which is particularly useful for the expression of antibodies as the product of interest, the membrane anchor is a transmembrane immunoglobulin anchor. Other suitable membrane anchors and preferred embodiments of a transmembrane immunoglobulin anchor are described in WO2007 / 131774, WO2005 / 073375 and PCT / EP2009 / 006246.

A host cell is also provided, which comprises at least:

(to)

an introduced polynucleotide encoding a product of interest;

(b)

an introduced polynucleotide encoding a first selection marker (sm I);

(C)

an introduced polynucleotide encoding a second selection marker (sm II), which differs from the first selection marker (sm I);

wherein the activity of the selection marker (sm I) or (sm II) is at least partially influenced by the activity of the other selection marker, where the selection markers (sm I) or (sm II) are involved in the folate metabolism and wherein the first selection marker (sm I) is a folate transporter and the second selection marker (sm II) is DHFR or a functional variant thereof.

According to one embodiment, the host cell comprises an expression vector or the combination of at least two expression vectors, as described in detail above and in the claims. The present inventors refer to the previous disclosure.

Additionally, the host cell may have at least one of the following characteristics:

The host cell is preferably a eukaryotic host cell. This eukaryotic cell is preferably selected from the group consisting of a mammalian cell, an insect cell, a plant cell and a fungus cell. Fungal cells and plant cells can be prototrophic for folates (that is, these cells can autonomously synthesize their own folates necessary for their cell viability, that is, for their cell growth and proliferation). The present invention particularly encompasses fungal and plant cells that are or may become auxotrophic for folates. This may be, for example, due to genetic manipulation, that is, the cells are now unable to synthesize sufficient amounts of the folates necessary for their cell viability. For example, the ability of fungal or plant cells to biosynthesize endogenously folates can be inactivated, for example, by an appropriate metabolic pathway, for example, by genetic alteration or genetic silencing of genes appropriate target or by inhibiting key enzymes, etc. Preferably, the host cell is a mammalian cell. This mammalian cell is preferably selected from the group consisting of a rodent cell, a human cell and a monkey cell. In particular, a rodent cell is preferred, which is preferably selected from the group consisting of a CHO cell, a BHK cell, an NS0 cell, a mouse 3T3 fibroblast cell and an SP2 / 0 cell. A more particularly preferred rodent cell is a CHO cell. A human cell is also preferred, which, preferably, is selected from the group consisting of a HEK293 cell, an MCF-7 cell,

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used in the prior art. In particular for the preferred combination of the use of a folate receptor in conjunction with a DHFR enzyme as selection markers (sm I) and (sm II), host cells having a higher productivity are obtained compared to the use of the markers of respective selection alone. Therefore, due to the higher stringency of the selection conditions, the selection procedure is optimized.

The selection method according to the present invention can also be carried out faster and more efficiently than conventional selection strategies known in the prior art, because selection for selection markers (sm I) and (sm II) in a selection step, if optimal cell culture conditions are used.

The method may additionally comprise a step of:

(c) select at least one host cell that expresses the product of interest with the desired yield.

The cells obtained as a result of the rigorous screening / selection process of the present invention in general will be isolated and can be enriched from the non-selected cells of the original cell population. They can be isolated and cultured as individual cells. However, it is also possible to use an enriched population. The host cells obtained can also be used in one or more additional selection rounds, optionally for additional qualitative or quantitative analysis or can be used, for example, in the development of a cell line for protein production. According to one embodiment, an enriched population of selected producing host cells as described above, is used directly as the population for the production of the polypeptide of interest in good yield.

Preferably, a host cell that stably expresses the product of interest is selected. The advantages of a stable transfection / expression are described in detail above. The present inventors refer to the previous disclosure.

In a very preferred embodiment of the method for selection, the plurality of host cells comprises the host cells according to the present invention, that is, as disclosed herein. We refer to the above detailed description, in particular to the appropriate selection markers (sm I) and (sm II) and combinations thereof.

Other preferred embodiments in particular with respect to the host cells and the expression vector, respectively, combination of expression vectors, are described in detail above. The present inventors refer to the previous disclosure.

The first selection marker (sm I) is a transporter / receptor that is capable of transporting folate into the host cell. This embodiment of the selection system according to the present invention does not require genomic suppression or attenuation of the alpha, beta or gamma endogenous folate receptor genes, prior to transfection and, therefore, can be applied to any receptor cell, even when some of the genetic expression of the endogenous folate receptor is present (see above). This key advantage is based on the fact that, following the transfection of the alpha folate receptor, cells can be exposed to abrupt and severe deprivation of folates (eg, folic acid) from the culture medium. Consequently, only transfectant cells that express significant amounts of the alpha folate receptor selection marker can carry enough folate to support DNA replication and cell proliferation. This occurs in the absence of any significant elevation in the expression of the endogenous folate receptor alpha gene. Additionally, this embodiment of the selection system according to the present invention does not suffer from loss of selection rigor due to the relief of selective pressure by means of an increase in the expression of alternative folate absorption routes, including the increase in the expression of the endogenous reduced folate carrier (RFC). This important advantage is due to the fact that, while the alpha folate receptor has an excess affinity for folic acid (Kd = 0.1 nM), the reduced folate carrier (RFC) exhibits an extremely poor affinity for acid folic (Km = 0.2-0.4 mM).

Since (sm II) is DHFR or a functional variant thereof, the selective culture medium additionally contains an inhibitor of a respective enzyme, for example, anti-folates, such as, for example, MTX, for the purpose of Provide selective conditions.

The selective culture medium that is used in at least one selection step may comprise one or more types of folate. The folate comprised in the selective culture medium in a limiting concentration is capable of being absorbed into and processed by the host cell. Folates and in particular folate derivatives that would not be processed by the host cell would not contribute to the selection pressure and, therefore, would not contribute to the limiting concentration. The selective culture medium may have one or more of the following characteristics:

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This DHFR / FolR-FACS Vector (VECTOR III) is compared to a DHFR-FACS reference vector that contains the neo gene as the second selection marker (VECTOR IV). VECTOR IV comprises the following main elements:

VECTOR IV ELEMENTS

CMV promoter / enhancer

Intron-RK

Polynucleotide encoding the antibody light chain

An SV40 Poly site

CMV promoter / enhancer

Intron-RK

Polynucleotide encoding the antibody heavy chain

Stop cod (filtered)

Polynucleotide encoding the immunoglobulin transmembrane anchor including the cytoplasmic domain

An SV40 Poly site

SV40 enhancer / promoter

Neomycin Resistance Gene

A synthetic Poly site

An ampicillin resistance gene

SV40 Promoter

Polynucleotide encoding DHFR (mutant)

SV40 intron fragment

An SV40 Poly site

Additionally, for co-transfection experiments using two expression vectors and the FACS classification, VECTOR V is created, which does not comprise any DHFR gene, but the neo gene and huFolR. VECTOR V comprises the following main elements:

VECTOR V ELEMENTS

CMV promoter / enhancer

Intron-RK

Polynucleotide encoding the antibody light chain

An SV40 Poly site

CMV promoter / enhancer

Intron-RK

Polynucleotide encoding the antibody heavy chain

Stop cod (filtered)

Polynucleotide encoding the immunoglobulin transmembrane anchor including the cytoplasmic domain

An SV40 Poly site

SV40 enhancer / promoter

Neomycin Resistance Gene

A synthetic Poly site

An ampicillin resistance gene

SV40 Promoter

huFolR

An SV40 Poly site

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Claims (1)

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